Evaluation of a bead-free immunoprecipitation technique coupled with tandem mass spectrometry for identification of plant-virus protein interactions

Authors: Deblasio, Stacy; BEREMAN, MICHAEL - North Carolina State University; MAHONEY, JACLYN - Boyce Thompson Institute; Thannhauser, Theodore - Ted; Gray, Stewart; MACCOSS, MICHAEL - University Of Washington; Heck, Michelle

Submitted to: Journal of Biomolecular Techniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/20/2017
Publication Date: 9/28/2017
Citation: DeBlasio, S.L., Bereman, M.S., Mahoney, J., Thannhauser, T.W., Gray, S.M., MacCoss, M.J., Heck, M.L. 2017. Evaluation of a bead-free immunoprecipitation technique coupled with tandem mass spectrometry for identification of plant-virus protein interactions. Journal of Biomolecular Techniques. 28(3):111-121. https://doi.org/10.7171/jbt.17-2803-002.
DOI: https://doi.org/10.7171/jbt.17-2803-002

Interpretive Summary: Plant viruses that infect agronomic crops cause severe economic hardship on growers around the world. Viruses that are spread by insect vectors are very difficult to control. Pesticides to control vector populations are routinely used in disease management strategies but they are ineffective in stopping the spread of a virus within a crop and are costly as well as harmful to the environment. Precision methods to block virus infection in plants or the spread of a virus by the insect vector represent new approaches to virus disease management and require an in depth knowledge of the protein interactions used by viruses in their plant hosts and aphid vectors. In this study, we evaluated the feasibility of using an inexpensive assay to identify plant proteins that interact with wild-type (WT) and mutant form of the Potato leafroll virus (PLRV) as these interactions exist within plant cells. PLRV is a virus that impacts potato production worldwide and is spread by the green peach aphid, Myzus persicae. Comparison of plant and viral proteins identified as interacting directly or indirectly with the virus using the inexpensive platform to those identified using traditional methods that are more comprehensive and also more costly, revealed that some plant-virus protein interactions can be reproducibly identified using the inexpensive assay. Our work provides labs with limited resources and those looking to do a cursory and inexpensive screen with a cheaper, easier alternative to characterizing interactions between plant and viral proteins.

Technical Abstract: Potato leafroll virus (PLRV) is an aphid-borne, positive sense, single stranded RNA virus in the Luteoviridae that causes significant loss to potato production worldwide. The capsid structure for this family consists of a non-enveloped, icosohedral shaped virion composed of two structural proteins, the coat protein (CP) making up the majority of the virion and a minor component known as the readthrough protein (RTP) that protrudes from the surface of the virion. Microdomains within each of these structural proteins regulate virus propagation and movement through specific interactions with host proteins. Protein immunoprecipitation (IP) coupled to high-resolution mass spectrometry provides a high-throughput approach in identifying and characterizing virus-host interactomes in a single experiment. Current methods rely on using viral-specific antibodies immoblized on agarose or magnetic beads to enrich for viral protein complexes from host homogenate. This technique, although well established, is fairly expensive and requires a large amount of antibody. In this study, we used an inexpensive, bead-free microplate assay coupled with tandem mass spectrometry to identify plant proteins that interact with wild-type (WT) and a DRTP mutant of PLRV in vivo. Comparison of host and viral proteins identified as enriched ³2-fold in both WT and mutant IPs using the bead-free method revealed a ~65% overlap with proteins identified as forming high-confident interactions with PLRV using antibody coated-magnetic beads with twenty of these proteins also detected as significant interactions in the beads-free assay. An additional nineteen high-confident virus-host interactions were only identified in the bead-free system. Two of these thirty-nine proteins, a 14-3-3 signal transduction protein and malate dehydrogenase 2, were identified as having a weakened or lost association with virus in the absence of the RTP in both immunoprecipitation assays, showing that the method is sensitive enough to detect quantitative differences between different, yet closely related viral bait proteins. Collectively, our analysis shows that the bead-free platform is an easy, low-cost alternative that can be used to reproducibly identify plant and viral proteins interacting with virion.